Facile synthesis of 4-O-β-N-Acetylchitooligosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone based on the transformation of chitooligosaccharide and its suppressive effects against the furylfuramide-induced SOS response

A facile synthesis method is described for transforming the reducing-end residue of chitooligosaccharides (DP 2-4) into lactone. The desired 4-O-β-N-acetylchitooligosyl lactones (GN(n)L) were conveniently prepared from chitooligosaccharides by consecutive dehydration and oxidation reactions to afford 4-O-β-tri-N-acetylchitotriosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN(3)L), 4-O-β-di-N-acetylchitobiosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN(2)L), and 4-O-β-2-acetamido-2-deoxy-D-glucopyranosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GNL). The resulting lactone derivatives exhibited considerable suppression (42.6-54.3% at a concentration of 400 μM) in umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2). Lactonization of the chitooligosaccharides was found to be essential for their suppression of the SOS-inducing activity.     

A sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM1011 having a high nitroreductase activity.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC'-'lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the beta-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.     

Facile Synthesis of 4-O-β-N-Acetylchitooligosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone Based on the Transformation of Chitooligosaccharide and Its Suppressive Effects against the Furylfuramide-Induced SOS Response

A facile synthesis method is described for transforming the reducing-end residue of chitooligosaccharides (DP 2–4) into lactone. The desired 4-O-β-N-acetylchitooligosyl lactones (GN_nL) were conveniently prepared from chitooligosaccharides by consecutive dehydration and oxidation reactions to afford 4-O-β-tri-N-acetylchitotriosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN₃L), 4-O-β-di-N-acetylchitobiosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN₂L), and 4-O-β-2-acetamido-2-deoxy-D-glucopyranosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GNL). The resulting lactone derivatives exhibited considerable suppression (42.6–54.3% at a concentration of 400 µM) in umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2). Lactonization of the chitooligosaccharides was found to be essential for their suppression of the SOS-inducing activity.     

Induction of umu gene expression in Salmonella typhimurium TA1535/pSK1002 by dimethyl sulfoxide (DMSO)

The genotoxicity of dimethyl sulfoxide (DMSO) was demonstrated by the umu test using Salmonella typhimurium TA1535/pSK1002 carrying the umuC-lacZ fusion gene. The level of beta-galactosidase activity which shows umu gene expression in the test system was dependent on the concentration of DMSO in the culture medium. The maximum beta-galactosidase activity was approximately 3.5 times as high as the background level with 10% of DMSO in the culture medium. The lowest concentration of DMSO required for a response of over twice the background level was approximately 5%. Four structurally related chemicals (acetone, di-n-butylsulfoxide, dimethylsulfide, methylphenylsulfoxide) did not show umu gene expression at their non-toxic doses.     

On the induction of umu gene expression in Salmonella typhimurium strain TA1535/pSK1002 by some nitrofurans.

Several nitrofurans were found to induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 as defined on the basis of at least a 2-fold increase of beta-galactosidase activity over the background level. beta-Galactosidase activity increased with increasing concentrations of the chemical, attained a maximum at a concentration which was different for different nitrofurans used, and then gradually decreased with a further increase of the nitrofuran concentration. The umu gene expression test revealed that the genotoxic activity was highest for furazolidone and lowest for 5-nitro-2-furaldehyde.     

Antimutagenic activity of flavonoids from Chrysanthemum morifolium

A methanol extract from the flower heads of Chrysanthemum morifolium showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract was re-extracted with hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction showed a suppressive effect. Suppressive compounds in the ethyl acetate fraction were isolated by silica gel column chromatography and identified as the flavonoids acacetin (1), apigenin (2), luteolin (3), and quercetin (4) by EI-MS, IR, and (1)H and 13C NMR spectroscopy. Compounds 1-4 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1-4 suppressed 60.2, 75.7, 90.0, and 66.6% of the SOS-inducing activity at a concentration of 0.70 micromol/ml. The ID50 (50% inhibitory dose) values of 1-4 were 0.62, 0.55, 0.44, and 0.59 micromol/ml. These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver-metabolizing enzymes. These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver-metabolizing enzymes, and UV irradiation. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S. typhimurium TA100.     

Suppressive effects of coffee on the SOS responses induced by UV and chemical mutagens

SOS-inducing activity of UV or chemical mutagens (AF-2, 4NQO and MNNG) was strongly suppressed by instant coffee in Salmonella typhimurium TA1535/pSK1002. As decaffeinated instant coffee showed a similarly strong suppressive effect, it would seem that caffeine, a known inhibitor of SOS responses, is not responsible for the effect observed. The suppression was also shown by freshly brewed coffee extracts. However, the suppression was absent in green coffee-bean extracts. These results suggest that coffee contains some substance(s) which, apart from caffeine, suppresses SOS-inducing activity of UV or chemical mutagens and that the suppressive substance(s) are produced by roasting coffee beans.     

Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens

The umu operon in Escherichia coli is responsible for chemical and radiation mutagenesis, and the expression of the operon itself is inducible by these DNA-damaging agents. The principle of the umu-test is based on the ability of the DNA-damaging agents, most of which are potential carcinogens, to induce the umu operon. A plasmid (pSK1002) carrying a fused gene umuC'-'lacZ was introduced into Salmonella typhimurium TA1535. The strain TA1535/pSK1002 enabled us to monitor the levels of umu operon expression by measuring the beta-galactosidase activity in the cells produced by the fusion gene. Using this strain, a simple, inexpensive, and sensitive system, the umu-test, for the screening of environmental mutagens and carcinogens was developed. 38 chemicals with different structures and modes of action, including 31 known animal carcinogens, were examined by the test to evaluate the system. The threshold sensitivity of the umu-test was approximately equal to that of the Ames test for chemicals genotoxic in both tests. By the umu-test, using the single tester strain, we detect many types of DNA-damaging agents for which the Ames test requires several tester strains. Furthermore, the umu-test provides a potential practical advantage for the screening of various environmental samples containing amino acids and nutrients such as urine, serum and foods.     

Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with β-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.     

Plasmid pSK1002-mediated mutator effect and SOS response and SOS mutagenesis of 2,5-dichloronitrobenzol in Salmonella typhimurium

The plasmid pSK1002 (umuC'-'lacZ) could increase the number of revertants induced by methyl methanesulfonate (MMS) and 4-nitroquinoline-N-oxide (4NQO) in Salmonella typhimurium TA 1535 (his-). The values induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were not different irrespective of the presence or absence of plasmid. However, the plasmid pKM101-mediated mutagenesis-enhancing effect was much greater than that mediated by pSK1002 as induced by the 3 mutagens mentioned above. Moreover, the plasmid pSK1002 could induced umu-mediated SOS response in the presence of any of these 3 mutagens or of mitomycin C, and a dose-response relationship was evident. It shows that pSK1002 (umuC'-'lacZ) has a dual biological effect, namely a mutator effect and the effect of inducing the SOS response. Besides, this study has proved SOS mutagenesis of 2,5-dichloronitrobenzol (2,5-DCNB) because of the dual indicator nature of pSK1002. Therefore, it is probable that pSK1002 could be further developed and applied in studying the relation between the SOS response and mutagenesis and in identifying environmental SOS mutagens.     

A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples.

The umu-microtest is a miniaturized automated short-term test version proposed for screening of umuC-dependent mutagenic potentials of chemicals relevant to environmental pollution, river water and industrial waste water. The test is based on the SOS/umu-test and has been modified in order to allow extensive testing of environmental samples. Genetically engineered Salmonella typhimurium (TA1535/pSK1002) are incubated on a microplate rotor in a sloping position for 2 h with the test samples, followed by addition of fresh culture medium to reach a 10-fold dilution of the incubation medium. 2 h later, the activity of the beta-galactosidase, which reflects umuC induction, is determined colorimetrically. The incubation of the bacteria in the presence of the test compounds as well as the assessment of beta-galactosidase activity takes place in 96-well microplates, thus enabling simultaneous screening of large numbers of samples. Data of the genotoxic potentials are available within 8 h. Computer-controlled automation is possible by using a laboratory workstation.     

Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/umu assay

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.     

Study on mutagenicity and antimutagenicity of BHT and its derivatives in a bacterial assay

The mutagenicity of butylated hydroxytoluene (BHT) and its derivatives was investigated by the Ames method using Salmonella typhimurium TA98 and TA100 with or without S9 mix. The compounds were not mutagenic in either indicator strain at concentrations ranging from 50 to 330 micrograms/plate (SQ: 3,5,3',5'-tetra-tert-butylstilbenequinone; VI-III: unidentified), 500 micrograms/plate (BE: 3,5,3',5'-tetra-tert-4,4'-dihydroxy-1,2-diphenylethylene; VI: 2,6-di-tert-butyl-4-methyl-4-tert-butylperoxy-2,5-cyclohexadienone ; VI-I: unidentified; VI-II: 3-acetyl-2,5-di-tert-cyclopenta-2,4-dienone) and 1000 micrograms/plate (BHT). The antimutagenic effects of BHT and its derivatives on mutagenesis by chemical agents were investigated in Salmonella typhimurium TA98 and TA100 and Escherichia coli WP-2 hcr-. VI-II suppressed the mutagenesis induced in TA98 and TA100 by 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and that induced in WP-2 hcr- by 4-nitroquinoline-1-oxide (4NQO) without decreasing cell viability. In WP-2 hcr-, the mutagenesis induced by AF-2 and ethyl methanesulfonate was also suppressed significantly. Mutations induced by methyl methanesulfonate were slightly inhibited. However, VI-II had no effect on the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Induction of SOS response in Salmonella typhimurium TA4107/pSK1002 by peroxynitrite-generating agent, N-morpholino sydnonimine

Salmonella typhimurium TA4107/pSK1002 strain was used to measure the SOS response induced by peroxynitrite. The parent strain TA4107 (oxydelta1[oxydelta(oxyR argH)1]) is sensitive to oxidative stress and the plasmid of pSK1002 carries a fused gene umuC'-'lacZ, in which umu and lacZ genes are involved in the induction of mutagenesis and beta-galactosidase activity, respectively. Therefore, the level of SOS response was monitored via beta-galactosidase activity. A bolus addition of authentic peroxynitrite (0.3-0.6 mM) increased about eight times the enzyme activity. In N-morpholino sydnonimine (SIN-1), which produces peroxynitrite from superoxide and nitric oxide generated through hydrolysis, addition of over 1mM SIN-1 induced four-five-fold activity. The SIN-1-induced SOS response was scarcely influenced by superoxide dismutase (SOD), catalase or a combination of both, removing the possibility of induction by superoxide, hydrogen peroxide and hydroxyl radical. Two types of peroxynitrite scavengers, mannitol (type I) and glutathione (type II), decreased the response. Mannitol showed a constant inhibition (70%) at a concentration up to 20 mM, exhibiting kinetics that are zero-order in mannitol and first-order in peroxynitrite. On the other hand, glutathione sharply reduced the response dependent on concentration up to 2 mM (90%), indicating second-order kinetics, first-order in both glutathione and peroxynitrite. Dihydrorhodamine (DHR)123, which traps peroxynitrite in a molar ratio of 1:1, efficiently inhibited the SOS response. These effects suggest that peroxynitrite, generated gradually from SIN-1, penetrates through the cell membrane, damages the DNA and induces the SOS response. This strain can thus, be used in screening of antioxidants against peroxynitrite-induced DNA damage in cells.     

The capacity of some nitro- and amino-heterocyclic sulfur compounds to induce base-pair substitutions

The capacity of 27 heterocyclic sulfur compounds to induce base-pair substitutions was investigated with Klebsiella pneumoniae ur- pro- and Salmonella typhimurium TA100 as test organisms. Among the compounds tested, all sulfur compounds with nitro groups and some thiazoles with an amino group were mutagenic. Among the nitrothiazoles, the most potent mutagen was niridazole, followed by 2-acetamido-5-nitrothiazole, 2-bromo-5-nitrothiazole, N-(5-nitrothiazol-2-yl)benzamide, and 2-amino-5-nitrothiazole. Of the nitrothiophenes, 2-nitrothiophene was more mutagenic than 3-nitrothiophene and 2,4-dinitrothiophene. 4-Nitroisothiazole was also mutagenic. Of the aminothiazoles, 2-amino-5-bromothiazole and 2-amino-5-chlorothiazole were mutagenic to both test organisms. With 2-amino-5-(p-nitrophenylsulfonyl)thiazole, a mutagenic action was only found with Salmonella typhimurium TA100, whereas 2-aminothiazole and 2-amino-4-methylthiazole were only mutagenic with Klebsiella pneumoniae. With the other 13 compounds, no mutagenic activity was observed. Of the coccidiostatics, 2-acetamido-5-nitrothiazole was also mutagenic on Escherichia coli K12 and Saccharomyces cerevisiae D4 but non-mutagenic on Salmonella typhimurium TA1530, TA1535, TA1537 and TA98, while 2-amino-5-nitrothiazole was mutagenic on Escherichia coli K12, Salmonella typhimurium TA1530, TA1535 and TA98, and non-mutagenic on strain TA1537 and on Saccharomyces cerevisiae D4.     

In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.     

Mutagenicity of the metabolites of the epoxide-diol pathway of safrole and its analogs. Study on Salmonella typhimurium]

Mutagenicity of the metabolites of the expoxide-diol pathway of safrole and analogues was studied on Ames' strains with Ames' method. Safrole, eugenol, eugenolmethylether, estragol, allylbenzene and 1'-hydroxysafrole, are not mutagen on TA 1535, TA 100 (point mutation) and TA 1537, TA 1538, TA 98 (frameshift mutations) without activation system. The corresponding epoxides that we have synthetized, are mutagens and inducers of point mutation in TA 1535 and TA 100. Dose-effect curves show differences between the mutagen efficiencies of these epoxides probably in relation with their electrophilic properties. On the other hand the 2', 3'-dihydro-2',3'-dihydroxisafrole was not mutagen in Ames' test. These results confirm the promutagen character of safrole and analogues and the role of the epoxides as proximate carcinogens.     

Antimutagenic activity of Terminalia chebula (myroblan) in Salmonella typhimurium.

Antimutagenicity of water and chloroform extracts of dried myroblan Terminalia chebula was determined against two direct acting mutagens, sodium azide and 4-nitro-o-phenylenediamine (NPD) in strains TA100 and TA1535, and TA97a and TA98 of Salmonella typhimurium respectively and S9-dependent mutagen 2-aminofluorene (2-AF) in TA97a, TA98 and TA100 strains. Water extract reduced NPD as well as 2-AF induced his+ revertants significantly but did not have any perceptible effect against sodium azide included his+ revertants in TA100 and TA1535 strains of S. typhimurium. The pre-incubation studies, where the extract was incubated at 37 degrees C for 30 min with the said mutagen prior to plating, enhanced the inhibitory effect. Autoclaving the water extract reduced the inhibitory effect but the reduction in the effect was not significant. No inhibitory effect was observed in any of the strains and against any of the test mutagens with chloroform extract.     

Chromosome breakage and neoplastic transformation of Syrian golden hamster embryonic cells in tissue culture by transplacental application of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

Hamster embryos were treated with 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) in vivo (in the mother) by transplacental application. The fetuses were isolated 24 h after administration of the AF-2 and cultured. Within the first 24h of primary culture, some parts of the cells were treated with colcemide for 3 h so that mitotic cells could be observed in the first cell cycle in vitro. Cultured embryonic fibroblasts in metaphase plates showed a marked dose-dependence in chromosomal aberrations. Transplacental application of AF-2 also caused slightly dose-dependent morphological transformation. When some transformed colonies were cloned and transferred to the hamster cheek pouch, these cells produced tumors in the host animals. This new in vivo--in vitro combination assay system is considered to be useful for detection of environmental potential carcinogens.     

Studies on the genotoxicity of endosulfan in bacterial systems

Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage lambda induction assay in WP2s (lambda) and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535/pSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage lambda in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage.